2-3% of the world's population is estimated to be infected with the hepatitis C virus (HCV), the etiological agent of non-A, non-B hepatitis. Most infections become chronic and can lead to cirrhosis, hepatocellular carcinoma, and liver failure. With no available vaccine or widely effective treatment, there is an urgent need for the development of antiviral therapies that control HCV replication. The core component of the HCV replication machinery is the NS5B RNA-dependent RNA polymerase, which catalyzes the synthesis of the single-stranded RNA HCV genome. NS5B is an attractive target for rational drug design, as polymerase inhibitors have been used successfully to control the replication of other pathogenic viruses. Recently, the interaction of NS5B with a host cell factor known as cyclophilin B(CyPB) was reported. CyPB was shown to stimulate RNA binding by NS5B, suggesting that it may be a cofactor important for efficient viral RNA synthesis. The experiments proposed here will test the hypothesis that CyPB directly stimulates RNA synthesis by the NS5B polymerase. A quantitative analysis of the effects of CyPB on the polymerase activities of recombinant NS5B will be performed. The effect of CyPB on NS5B from several different HCV strains will be compared, including a recently discovered strain of HCV that behaves differently from most other strains, both clinically and, in laboratory experiments, in its response to cyclophilin B. The basis for any observed differences will be determined by mutational analysis both in vitro and in cell culture. Because CyPB is inhibited by the widely used immunosuppressive drug cyclosporin A, these studies could impact drug regimens for many liver transplant patients, the majority of whom suffered liver failure due to HCV infection. The impact of CyPB on NS5B inhibitors will also be examined. PUBLIC HEALTH RELEVANCE: Hepatitis C is recognized as a global health crisis, with approximately one in fifty people currently infected with the hepatitis C virus (HCV). The experiments proposed here would improve our understanding of the mechanism of HCV replication, which in turn should aid the development of more widely effective antiviral drugs.